The current paradigm for evolution of arthropod-borne viruses (arboviruses) is the trade-off hypothesis, which proposes that cycling between different vertebrate and invertebrate hosts, presents significant constraints on genetic change of arboviruses. Studying these constraints in mosquito-borne viruses has led to a new understanding of epizootics and virus emergence. However, the trade-off hypothesis was based on mosquito- borne viruses and is assumed to be correct for tick-borne viruses too. This represents a critical gap in the field because the transmission cycles of tick-borne versus mosquito-borne viruses differ in key features that play a major role in virus perpetuation, such as vertical and transstadial transmission. Among the tick-borne viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread cause of severe and fatal human disease with thousands of cases annually. The NIAID has recently promoted CCHFV to a Category A priority pathogen due to the lack of vaccines, high mortality rates, and potential for major public health impact. Compared to other arboviruses, CCHFV shows an extraordinary amount of genetic diversity among strains. Its diversity has been linked to increased virulence in humans and emergence in new environments and impedes diagnostic tool and countermeasure development. We recently established the first experimental transmission model for CCHFV in BSL4 by utilizing Hyalomma marginatum ticks (the main vector) and mice. By using next generation sequencing, we detected a significant number of non-synonymous mutations in CCHFV recovered from ticks after only a single transstadial transmission. The high mutation frequency was not detected in CCHFV obtained from the mammalian host. These findings are in contradiction to the trade-off hypothesis and suggested a greater selection pressure for CCHFV in the tick than previously expected. Given these data, we hypothesize that transstadial transmission by the tick vector is a major determinant of the genetic diversity of CCHFV, and the molting phase is the key process responsible for expanding CCHFV intra-host diversity. Aim 1 will analyze CCHFV mutations and viral diversity after multiple transstadial transmissions and transmission to the mammalian host to characterize the CCHFV selection pressure over time. Aim 2 will track CCHFV dissemination in the tick during molting to identify potential viral expansion hotspots and understand how transstadial transmission leads to greater intra-host diversity. The information generated from these studies will give us a new understanding of the tick-virus-host interaction that shapes the genomic diversity of this emerging virus, and will lead to a reevaluation of the trade-off hypothesis from a tick-borne virus perspective. Characterizing the forces that shape CCHFV genetic plasticity will be important in the understanding of virus adaptations to changing environments and in determining viral evasion of the vector and host immune response.